摘要
目的:构建人血管内皮生长因子165(hVEGF165)的真核表达质粒pIRES2-EGFP-hVEGF165,通过脂质体法将其转入人胎盘源间充质干细胞(HPMSCs)中,鉴定hVEGF165的表达活性及携带目的基因的HPMSCs的多向分化潜能。方法:采用逆转录聚合酶链式反应(RT-PCR)技术,从人白血病细胞HL-60中扩增出hVEGF165的基因片段,构建pIRES2-EGFP-hVEGF165重组质粒,经酶切检测其构建的正确性,通过脂质体法转染HPMSCs后分别采用RT-PCR、Western blotting法及MTT法检测其转录和表达活性;荧光显微镜下观察增强型绿色荧光蛋白(EGFP),检测含有报告基因EGFP空载体及携带报告基因和目的基因的真核表达载体转染靶细胞的情况;并再次鉴定转染后HPMSCs的多向分化潜能。结果:成功构建重组质粒pIRES2-EGFP-hVEGF165;RT-PCR、Western blotting及MTT法检测表明,构建的质粒pIRES2-EGFP-hVEGF165具有转录及表达活性;绿色荧光显微镜下观察到EGFP表达,目的基因成功转入靶细胞,携带目的基因的HPMSCs仍保持多向分化潜能。结论:成功构建具有表达活性的hVEGF165真核表达质粒pIRES2-EGFP-hVEGF165,hVEGF165在HPMSCs中具有转录及表达活性,且对HPMSCs的增殖具有促进作用;HPMSCs本身可能具有hVEGF165的内分泌功能。
Objective To construct the eukaryotic expression plasmid pIRES2-EGFP-hVEGF165 of human vascular endothelial growth factor 165（hVEGF165）,and to transfect pIRES2-EGFP-hVEGF165 into human placental mesenchymal stem cells（HPMSCs） through liposome,and to identify the express activity of hVEGF165 and the multi differentiation potential of HPMSCs carrying the target gene.Methods From human leukemia cells HL-60 the gene fragment of hVEGF165 was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR）,the pIRES2-EGFP-hVEGF165 recombinant plasmid was constructed,and its correctness was detected by restriction enzyme digestion;the activity of transcription and expression of HPMSCs transfected by liposome were respectively detected with RT-PCR,Western blotting and MTT assay;the enhanced green fluorescence protein（EGFP） was observed under fluorescence microscope;and the conditions of transfected target cells of empty vector containing reporter gene EGFP,eukaryotic expression vector carrying reporter gene and target gene were detected;and the multi-differentiation potential of HPMSCs after transfection was indentified.Results The recombinant plasmid pIRES2-EGFP-hVEGF165 was successfully constructed through enzyme cutting identification.The constructed plasmid of HPMSCs after transfection with pIRES2-EGFP-hVEGF165 had transcription and expression activities;the EGFP expression was detected under fluorescence microscope,and the target gene was successfully transferred into target cells,and the HPMSCs carrying the target gene still maintained multipotentiality.Conclusion The eukaryotic expression plasmid pIRES2-EGFP-hVEGF165 carrying hVEGF165 with expression activity is successfully constructed,and the plasmid is successfully transferred into HPMSCs.The hVEGF165 in HPMSCs has the activities of transcription and expression,and hVEGF165 could promote the proliferation of HPMSCs,and the HPMSCs itself might have the endocrine function of hVEGF165.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2012年第2期270-275,I0003,共7页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅青年科技基金资助课题(20091090)
吉林大学基本科研业务经费资助课题(450060323446)
江苏省镇江市社会发展科技支撑项目资助课题(SH2010043)
关键词
人血管内皮生长因子165
人胎盘源间充质干细胞
脂质体
转染
human vascular endothelial growth factor 165
human placental mesenchymal stem cells
liposome
transfection
